Mitochondrial DNA from paramecia replicates via lariat and dimer length molecule intermediates. Dimer length molecules are processed to two monomer length linear molecules and replication is initiated by closure of one end. Further, 13 species of Paramecia are available for analysis and we have shown that each of the five studied has a distinctive endonuclease fragmentation pattern. We propose to construct a detailed restriction map of at least two species so as to determine structural and functional relationships. In addition, we will determine the localization of specific genes, ribosomal RNA, on this map as well as the nucleotide sequence of the initiation site. The sequence analysis work will be facilitated by cloning fragments isolated from EcoRI and Bam HI digestion. DNA isolated from these clones can also be used for future studies in interspecies homology. It is hoped that these studies will provide fundamental information on the mechanism of replication of linear genomes as well as offer insight into mitochondrial function, biogenesis and evolution.